外泌体在体外对乳腺癌细胞耐药信息传递的作用

发布时间：2018-06-20 06:02

本文选题：外泌体 + 乳腺癌　；参考：《西安交通大学学报(医学版)》2017年02期

【摘要】：目的探讨外泌体(exosomes,EXOs)在乳腺癌细胞间耐药信息的传递作用。方法选用乳腺癌敏感细胞株(MCF-7)及其阿霉素耐药株(MCF-7/ADR)为模型,用超速离心法提取MCF-7/ADR外泌体(ADR/EXO);用透射电子显微镜观察鉴定细胞外泌体形态;运用倒置荧光显微镜观察ADR/EXO与MCF-7细胞的相互作用过程;运用激光共聚焦显微镜观察MCF-7与MCF-7/ADR在Transwell小室共培养60h后细胞内阿霉素药物浓度变化;运用CCK-8法检测ADR/EXO与MCF-7细胞共培养后对阿霉素的IC50的影响;运用RT-PCR检测MCF-7与ADR/EXO共培养后MDR1基因表达水平的变化;运用Western blot法检测MCF-7与ADR/EXO共培养12h和72h后P-gp表达水平的变化。结果透射电子显微镜下,外泌体呈现典型的圆形或椭圆形杯口状结构,直径为40~100nm;用PKH67标记ADR/EXO显示,ADR/EXO是可以被MCF-7细胞摄取;与ADR/EXO共培养后敏感细胞株MCF-7对阿霉素的IC50耐药性升高2.62倍,差异有统计学意义(P0.05);经过与MCF-7/ADR在Transwell小室中共培养60h后的MCF-7细胞,胞内可见阿霉素聚集减少,核区分布减少,荧光强度减弱;MCF-7+ADR/EXO组MDR1的mRNA表达量明显增加,是MCF-7组的8.69倍,差异有统计学意义(P0.05);与MCF-7组相比,共培养12h的MCF-7+ADR/EXO组P-gp相对表达量升高4.64倍,差异有统计学意义(P0.05);与MCF-7组相比,共培养72h的MCF-7+ADR/EXO组P-gp相对表达量升高3.37倍,差异有统计学意义(P0.05)。结论乳腺癌外泌体可能具有传递耐药信息的作用,耐药细胞外泌体能够使敏感细胞获得一定的耐药性,耐药细胞外泌体向敏感细胞传递P-gp,不仅可以通过蛋白形式进行传递,而且可以通过mRNA的形式进行传递。
[Abstract]:Objective to investigate the role of exosomesus EXOsin in the transmission of drug resistance information between breast cancer cells. Methods MCF-7) and its adriamycin resistant cell line MCF-7 / ADR) were used as models, and ADR / EXOO was extracted by ultracentrifugation, and the morphology of exocrine of MCF-7 / ADR was observed and identified by transmission electron microscope (TEM). The interaction between ADR / EXO and MCF-7 cells was observed by inverted fluorescence microscope, and the concentration of adriamycin in MCF-7 / MCF-7 / ADR cells was observed by laser confocal microscopy after co-cultured in Transwell chamber for 60 hours. The effect of co-culture of ADR- / EXO and MCF-7 cells on the IC50 of adriamycin was detected by CCK-8 method, the expression of MDR1 gene was detected by RT-PCR after co-culture of MCF-7 and ADR- r-EXO, and the expression of P-gp was detected by Western blot assay after co-culture of MCF-7 and ADRP-EXO for 12h and 72h. Results under the transmission electron microscope, the exocrine presented a typical round or oval cup shaped structure with a diameter of 40,100nm.The PKH67 labeled ADR / EXO showed that ADR / EXO could be absorbed by MCF-7 cells. After co-culture with ADR / EXO, the IC50 resistance of MCF-7 to adriamycin increased by 2.62 times, the difference was statistically significant (P 0.05). After co-cultured with MCF-7 / ADR in Transwell chamber for 60 hours, the intracellular concentration of doxorubicin decreased and the distribution of nuclear region decreased. The expression of MDR1 mRNA in MCF-7 ADR / EXO group was 8.69 times higher than that in MCF-7 group, the difference was statistically significant (P 0.05), compared with MCF-7 group, the relative expression of P-gp in MCF-7 ADR / EXO group was 4.64 times higher than that in MCF-7 group, and compared with MCF-7 group, the difference was statistically significant (P 0.05), compared with MCF-7 group, the expression of P-gp in MCF-7 ADR / EXO group was 4.64 times higher than that in MCF-7 group. The relative expression of P-gp in MCF-7 ADR / EXO group was 3.37 times higher than that in MCF-7 ADR / EXO group for 72 h, and the difference was statistically significant (P 0.05). Conclusion the exosomes of breast cancer may transmit the information of drug resistance. The exosomes of drug-resistant cells can make the sensitive cells obtain certain drug resistance. The exocrine of drug-resistant cells can transfer P-gpto sensitive cells, not only through the form of protein. And it can be transmitted in the form of mRNA.
【作者单位】：滨州医学院附属医院甲状腺乳腺外科;
【基金】：国家自然科学基金资助项目(No.81173601、30972932) 滨州市科技发展计划项目(No.2015ZC0301) 滨州医学院科研计划与科研启动基金(No.BY2014KYQD36) 山东省高等学校科技计划项目(No.J15LL51)~~
【分类号】：R737.9

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